Astrocytic RNA editing regulates the host immune response to alpha-synuclein

Abstract

RNA editing is a posttranscriptional mechanism that targets changes in RNA transcripts to modulate innate immune responses. We report the role of astrocyte-specific, ADAR1-mediated RNA editing in neuroinflammation in Parkinson's disease (PD). We generated human induced pluripotent stem cell-derived astrocytes, neurons and cocultures and exposed them to small soluble alpha-synuclein aggregates. Oligomeric alpha-synuclein triggered an inflammatory glial state associated with Toll-like receptor activation, viral responses, and cytokine secretion. This reactive state resulted in loss of neurosupportive functions and the induction of neuronal toxicity. Notably, interferon response pathways were activated leading to up-regulation and isoform switching of the RNA deaminase enzyme, ADAR1. ADAR1 mediates A-to-I RNA editing, and increases in RNA editing were observed in inflammatory pathways in cells, as well as in postmortem human PD brain. Aberrant, or dysregulated, ADAR1 responses and RNA editing may lead to sustained inflammatory reactive states in astrocytes triggered by alpha-synuclein aggregation, and this may drive the neuroinflammatory cascade in Parkinson's.

Fig. 1.. Schematic illustrations of experimental paradigm and the differentiation protocols.

(A) hiPSC-derived from five healthy individuals are differentiated into astrocytes (in-house lines: C1, C2, and C3; commercial lines from ACS: C4 and C5) and from one individual into neurons. (B) Differentiation of cortical region–specific astrocytes was performed, modifying established protocols (78, 79). NPCs using an established protocol [Shi et al., 2012 (29)] were differentiated into either neurons or GPC (glial precursor cells) after 30 days from NPC and then further differentiated into mature astrocytes. (C) Immunocytochemistry images show that three lines of hiPSC-derived astrocytes express the astrocytic marker, GFAP. (D and E) There is a calcium response to ATP in iPSC-derived astrocytes. Representative traces of calcium (D) and the percentage of cells (E) in response to ATP. (F) hiPSC-derived astrocyte enables uptake of glutamate (Glutamate Assay Kit, ab83389/K629-100, Abcam). (G and H) Composition of hiPSC-derived neuron and astrocyte coculture assessed using MAP2 (neuronal marker) and GFAP (astrocytic marker) immunocytochemistry together with representative images of a neuronal, astrocyte, and coculture (G) and the quantification (H). (I) Uniform Manifold Approximation and Projection (UMAP) plot showing the clustering of the integrated dataset using the cells from all the samples (basal and αsyn-O–treated astrocytes, neurons, and coculture samples). (J) Dot plots showing the expression of the astrocyte and neuron marker genes in the clusters identified in the single-cell RNA-seq data. (K) UMAP overlaid with the correlation coefficients, showing the correlation of the two astrocyte clusters with the astrocytes from Leng et al. (27). (L) Gene Ontology (GO) terms associated with the genes up-regulated in astrocyte clusters 1 and 2. (A) and (B) were created with BioRender (https://biorender.com/)

Fig. 2.. Oligomer treatment of astrocytes induces an inflammatory state.

(A) Uptake of monomeric species of labeled α-syn (A53T monomer) in astrocytes and neurons (yellow and red dotted lines, respectively) was detected, and formation of oligomers inside cells confirmed using FRET. (B and C) No difference in the formation of oligomeric species observed despite higher uptake of total α-syn in astrocytes compared to neurons (n = 3 independent inductions). (D and E) Application of αsyn-O induces overproduction of ROS. (F and G) Cell death induced by αsyn-O detected in astrocytes at low levels. (H) Cytokine release in αsyn-O–treated astrocytes is time dependent with TNF-α level highest after 24 hours of incubation with αsyn-O. (I) Morphological changes were traced using Fluo4 across the same time course as the cytokine release. (J) αsyn-O–treated astrocytes induce an inflammatory state by releasing a range of cytokines (basal, αsyn-O treated for interleukin-13 (IL-13), 3.02 ± 0.04 and 5.62 ± 0.05 pg/ml; IL-6, 3.11 ± 0.13 and 23.8 ± 2.4 pg/ml; IL-8, 79.11 ± 18.07 and 152.01 ± 7.29 pg/ml; IL-1B, 0.955 ± 0.03011 and 3.38 ± 0.0772; INF-γ, 0.51 ± 0.11 and 2.05 ± 0.1 pg/ml; TNF-α, 0.33 ± 0.0414 and 4.16 ± 0.187). (K) Cell-type proportions (excluding cell types with proportions <1%) for each of the cell types in the bulk astrocyte samples, basal, and αsyn-O treated, predicted by Scaden. (L) Top 10 GO terms associated with up- and down-regulated differentially expressed genes at FDR < 5% and ≥ twofold change in expression in the astrocytes basally versus with αsyn-O treatment. a.u., arbitrary unit.*P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3.. Coculture setting provides evidence of the astrocytes becoming more inflammatory.

(A) Effect of αsyn-O on cytokine release in the cocultures (basal IL-13: 3.091 ± 0.0139 pg/ml and αsyn-O treated 5.88 ± 0.238 pg/ml; IL-6: basal 2.75 ± 0.00174 pg/ml, αsyn-O treated 10.026 ± 0.0593 pg/ml; IL-8: basal 71.09 ± 6.107 pg/ml, αsyn-O treated 257.2 ± 1.58 pg/ml; IL-1B: basal: 0.231 ± 0.0835 pg/ml, αsyn-O treated 2.99 ± 0.381 pg/ml; INF-γ: basal 0.392 ± 0.0329, αsyn-O treated 2.078 ± 0.180 pg/ml; TNF-α: basal 0.392 ± 0.0329, αsyn-O treated 2.078 ± 0.1801) (B to D) αsyn-O induces toxicity in neurons alone and in coculture with astrocytes and induces increased levels of ROS in neurons alone and in coculture. (E) Plot showing the cell-type proportions estimated by Scaden from the single-cell data in the coculture samples, basally (lighter shade) and with αsyn-O treatment (darker shade). There is a significant difference between the cell-type proportions of astrocyte cluster 1 and 2 in the coculture with αsyn-O treated compared to basal. (F) GO terms associated with the up- and down-regulated differentially expressed genes with at least ≥twofold change in expression in the coculture αsyn-O treated versus basal. (G) Visualizing gene ratios of the enrichments observed in the up-regulated differentially expressed genes of the astrocytes (cyan) compared to that of the coculture (blue) (*P < 0.05, **P < 0.01, ***P < 0.001).

Fig. 4.. Type 1 IFN response leads to activation of ADAR.

(A) Model summarizing the role of ADAR in regulating the innate immune response to dsRNA with the log₂(Fold change) of the genes that are significantly differentially expressed (at FDR < 5%) in the astrocytes on αsyn-O treatment. Model created with BioRender (https://biorender.com/). (B) Plot showing the significantly differentially spliced junctions in ADAR in the astrocytes on αsyn-O treatment, with the transcripts and protein isoforms. (Coculture, fig. S5, A and B).

Fig. 5.. ADAR induced in A-to-I editing in astrocytes and cocultures with αsyn-O treatment.

(A) Number of editing sites in each sample, showing genomic locations. (B) Number of differentially edited sites when comparing αsyn-O treated to samples basally in each culture. X axis shows the decreased editing (blue, including lost) and increased editing (pink, including new). (C) Change in editing rate in basal versus αsyn-O treated samples in each culture. (D) Consequences of editing sites at baseline, and in differentially decreased or increased sites across cultures. (E) GO terms associated with the significantly differentially edited and differentially expressed (FDR < 5% and FC ≥ 2) in the astrocytes and coculture αsyn-O, treated versus basal. (F) Expression of ADAR and ADARB1 in single nuclear postmortem brain RNA. Top, shows expression by cell type, and the bottom shows expression within astrocytic clusters. Astrocyte cluster A is defined by expression of ADGRV1 and SLC1A2; astrocyte cluster B by GFAP, S100B, and AQP4; and astrocyte cluster C by VIM, SOX9, and FOS. (G) Mean residuals per editing site, after correction for covariates, in controls and diseased PD postmortem brain samples. (H) Expression weighted cell-type enrichment analysis of genes with 50 greatest increases in mean editing rate. Abbreviations: EN, excitatory neurons. IN, inhibitory neurons. OPC, oligodendrocyte progenitor cells.